The overall goal of this proposal is to understand the calcium (Ca2+)-dependent stimulus-secretion coupling mechanisms that regulate beta-cell function in vivo from the perspectives of biophysics, physiology and molecular biology. This goal will be achieved by studying intracellular Ca 2+ concentration, mitochondrial function, glucose metabolism, and insulin secretion in mouse and human islets in which the beta-cells have been engineered specifically for functional imaging. Ca2+-dependent signal transduction mechanisms that regulate insulin secretion have been well-defined in vitro using intact islets, primary cultures of beta-cells, and insulinoma cells. Notwithstanding the important contributions of the in vitro approaches, knowledge of the mechanisms underlying beta-cell function in intact islets in vivo remains incomplete. Several technical limitations of the current imaging methodologies do not permit the study of beta-cell function within the complex multicellular environment of the pancreas in situ. The central focus of the experiments described in this proposal is to develop and characterize a novel imaging approach, which will facilitate the study of islet cell function in situ. In Specific Aim 1, viral gene transfer vectors will be used to transduce mouse cells with genetically targeted biosynthetic fluorescent Ca 2+ sensors. Their effectiveness in studying a-cell biophysical and physiological responses following secretagogue stimulation will be evaluated. In Specific Aim 2, the utility of these sensors as functional imaging indicators in intact mouse islets will be assessed. In Specific Aim 3, beta-cells within intact human islets will be engineered to express Ca 2+ biosensors and human islet function studied in vitro with comparison to mouse islets. In Specific Aim 4, transgenic mouse models in which the beta-cells have been genetically enhanced to express Ca 2+ biosensors will be developed and characterized in vitro and in vivo by confocal microfluorometry and by measurements of insulin secretion. The studies will provide new understanding of islet cell biology that will benefit clinical strategies to preserve and maintain functional beta-cell mass.